Combination vaccine for swine dysentery and method of use

ABSTRACT

A combination vaccine for increasing the resistance of swine to dysentery infection comprises killed cells of a virulent isolate of Treponema hyodysenteriae in combination with concentrated killed cells of Bacteroides vulgatus, or Fusobacterium necrophorum, or mixtures thereof. This combination vaccine can be adapted for either oral or parenteral administration. For parenteral administration preferably only Bacteroides vulgatus is used in combination with Treponema hyodysenteriae. The oral vaccine is enteric-coated.

BACKGROUND AND PRIOR ART

An anaerobic spriochete, Treponema hyodysenteriae, has beencharacterized as the primary etiological agent in swine dysentery.Harris, D. L.; Glock, R. D.; Christensen, C. R.; and Kinyon, J. M.: Vet.Med./Small Animal Clin. 67:61 (1972); Taylor, D. J.; and Alexander, T.J. L.: Brit. Vet. J. 127:108 (1971). But relatively little is knownabout the immunology of swine dysentery although resistance toreinfection can be demonstrated in convalescent pigs. In 1976, Glock etal reported that parenteral vaccination of pigs with killed cells of avirulent isolate of T. hyodysenteriae provided a significant degree ofprotection against subsequent intragastric challenge with live virulentT. hyodysenteriae. Glock, R. D., Schwartz, K. J., and Harris, D. L.,Proceedings, International Pig Veterinary Society Congress, June 1976,Ames, Iowa. The vaccine was given in six intravenous injections at 6-dayintervals. This was the first reported success in immunizing swineagainst swine dysentery infection.

Hudson et al found that oral dosing of an attenuated strain of T.hyodysenteriae provided no protection against subsequent challenge.Hudson, M. J., Alexander, T. J. L., Lysons, R. J., Wellstead, P. D.,Brit. Vet. J. (1974) 130:37. Subsequently, Hudson et al attempted toimmunize pigs with live attenuated T. hyodysenteriae using a combinationof oral dosing and parenteral inoculation. Hudson, M. J., Alexander, T.J. L., Lysons, R. J., Prescott, J. F., Res. Vet. Science (1976) 21:366.Oral doses were administered on three consecutive days, and after aninterval of several days, intraperitoneal vaccinations wereadministered, which were followed after several more days withintramuscular vaccinations. The overall results of these tests weresummarized as follows: "Although vaccination appeared to enhanceimmunity to swine dysentery, half of the vaccinated pigs developed thedisease. This level of protection would be unlikely to be of practicalvalue in the field."

Treponema hyodysenteriae is a true pathogen in the sense that inconventional pigs Koch's postulates have been fulfilled. Glock, R. D.,and Harris, D. L., Vet. Med./Small Animal Clin. 67 (1972): 65-68;Kinyon, J. M., Harris, D. L. and Glock, R. D., Infect. Immun. 15 (1977):638-646. The status of T. hyodysenteriae as a pathogen is furtherevidenced by the fact that herds of pigs exist which are free of swinedysentery and are also free of T. hyodysenteriae. Kinyon, J. M., Songer,J. G., Janc, M., and Harris, D. L., In Proceedings 19th Annual Amer.Assoc. of Vet. Lab. Diag. (1976): 65-74. By contrast, pigs from thesesame herds will develop signs and lesions of swine dysentery when orallyinoculated with only T. hyodysenteriae. Kinyon, J. M., Harris, D. L.,and Glock, R. D., Infec. and Immun. 15 (2), (1977): 638-646. However, T.hyodysenteriae appears to require elements of the normal colonic florato exert its pathogenicity.

While swine dysentery infection can be reproduced in field-raised pigsby dosing them orally with pure cultures of Treponema hyodysenteriae,attempts to do so have failed until recently. In 1975, Meyer et alreported that swine dysentery was produced in gnotobiotic piglets bydosing them orally with T. hyodysenteriae together with 4 gram negativenon-sporing rod-shaped anaerobes. Meyer, R. C., Simon, J., and Byerly,C. S., Vet. Path. 12, (1975): 45-54. The anaerobes were not specificallyidentified by Meyer et al, but they were tentatively designated aseither species of Bacteroides or Fusobacteria. In further tests withgnotobiotic pigs, Harris et al found that lesions typical of acute swinedysentery were produced by intragastric inoculation of T. hyodysenteriaein combination with Bacteroides vulgatus, either alone or withFusobacterium necrophorum. All bacteria were administered live. Harris,D. L., Alexander, T. J. L., Whipp, S. C., Robinson, I. M., Glock R. D.,and Matthews, P. J., J. Amer. Vet. Med. Assoc. 172 (1978): 468-471. Infield raised pigs, it would be expected that natural colonic flora wouldinclude B. vulgatus, F. necrophorum and similar bacteria so that T.hyodysenteriae can exert its pathogenicity, producing the swinedysentery infection.

SUMMARY OF INVENTION

Swine dysentery vaccines containing a combination of antigens preparedfrom Treponema hyodysenteriae and Bacteroides vulgatus have an enhancedaction in increasing the resistance of swine to dysentery infection.More specifically, the combination vaccines of this invention containconcentrated killed cells of a virulent isolate of Treponemahyodysenteriae in combination with concentrated killed cells ofBacterioides vulgatus. From 0.25 to 2 parts by weight (dry basis) of theBacteroides vulgatus cells should be present per part of the T.hyodysenteriae cells, and preferably from 0.5 to 1.5 parts ofBacteroides vulgatus cells per part of T. hyodysenteriae. Thiscombination can be used in either an oral or a parenteral vaccine, andmay be further adapted for intramuscular injection by including asuitable adjuvant.

For use as an oral vaccine, Fusobacterium necrophorum in the form ofconcentrated killed cells can be employed instead of or in addition toBacteroides vulgatus. One preferred oral vaccine contains bothBacteroides vulgatus and Fusobacterium necrophorum cells with the T.hyodysenteriae antigen, and each are used in the relative proportions toT. hyodysenteriae specified above with respect to B. vulgatus.

The oral preparation is used in enteric-coated form. The enteric coatingis selected so that it is resistant to dissolving in the swine stomachwhile dissolving in the swine intestines to release the antigenic cellsfor immunizing action. The enteric-coated oral vaccine may be used inconjunction with a parenteral vaccine prepared in accordance with thepresent invention. However, the combination oral and parenteral vaccinesof this invention can also be employed separately.

For the most effective results, it is important to administer relativelylarge doses of the vaccines. For example, on the basis of the T.hyodysenteriae content at least 2 milligrams (dry basis) should beadministered per parenteral dose and at least 3 mg. per oral dose.Similar amounts are preferably used when the animals are given bothparenteral and oral inoculations. Both the parenteral and oral doses arepreferably repeated. For example, two parenteral doses can be given withseveral days intervening between doses, and the oral doses can berepeated over several consecutive days.

DETAILED DESCRIPTION

The present invention can be practiced with any virulent isolate of T.hyodysenteriae. Attenuated or non-virulent isolates or strains are notdesirable. A virulent isolate or strain is one which is capable ofproducing a typical swine dysentery infection. One suitable isolate hasheretofore been identified in the literature as B204. See Kinyon, J. M.,and Harris, D. L.: Vet. Rec. (1974): 95:219. Referred to in the samepublication is the isolate identified as B234, which can also be used inpracticing the present invention. However, type strain B78 (ATCC No.27164) is not suitable, being non-virulent. Isolates B204 and B234 havebeen deposited with the American Type Culture Collection; B204 beingidentified as ATCC No. 31212 and B234 as ATCC No. 31287. It should beunderstood that these isolates are representative of class of virulentisolates or strains which can be employed.

The T. hyodysenteriae cells for preparation of the vaccines can becultured using trypticase soy broth (TSB) with 10% (v/v) fetal calfserum (FCS). For example, the inoculated broth can be incubated at37°-38° C. under an anaerobic atmosphere, such as 50:50 H₂ :CO₂ or CO₂alone. The gaseous atmosphere should be deoxygenated. For furtherdetails, see Kinyon, J. M., and Harris, D. L.: Vet. Rec. (1974): 95:219.

For preparing vaccines in accordance with the present invention, it isbelieved that any strain or isolate of Bacteroides vulgatus orFusobacterium necrophorum can be used. Strains of these bacteria whichcan be used in practicing the present invention are therefore readilyavailable, such as those on deposit with the American Type CultureCollection, Rockville, Md. For B. vulgatus these include the type strainATCC No. 8482, and other strains or isolates, such as ATCC No. 31376.

For F. necrophorum the available strains include the ATCC referencestrain No. 25286, and the strain identified by ATCC No. 27852. A strainof B. vulgatus employed in some of the experiments leading to thepresent invention, is identified as strain 28, and has been depositedwith the American Type Culture Collection under ATCC No. 31376. Sinceneither Bacteroides vulgatus or Fusobacterium necrophorum are specificpathogens, there appears to be no difference for the purpose of thepresent invention between freshly isolated strains, and those which havebeen cultured in vitro for a large number of passages. However, if thesynergistic antigenic action should be found to be lessened afterprolonged in vitro cultivation, original cultures of the strains can beused, or freshly isolated strains can be obtained from the intestines ofswine, where both B. vulgatus and F. necrophorum are normally present infield-raised pigs.

Standard media and culture techniques can be used for growing the B.vulgatus and F. necrophorum. For example, each bacterium can be grownseparately in a standard pre-reduced anaerobically sterilizedpeptone-yeast extract media with added glucose. For example, 0.5%glucose can be added to the peptone-yeast broth. See Holdeman, L. V.,Cato, E. P., and Moore, W. E. C.: Anaerobe Laboratory Manual, pp.141-148, V.P.I. Anaerobe Laboratory, Blacksburg, Va. (4th ed. 1977). Thecultures may be incubated under deoxygenated CO₂ at 37° C.

After the fermentation has been completed, the cells can be recoveredand concentrated by centrifugation or ultrafiltration to obtain a cellslurry for further processing. The cells are killed by a suitableprocedure, either in the fermenter or after recovery. Standard killingagents may be used such as formalin or merthiolate. For example, akilling-concentration of formalin, such as 0.2% formalin (v/v), can beadded to the fermenter or to concentrated cell slurry. The killed cellsof T. hyodysenteriae, B. vulgatus, and F. necrophorum are used toprepare the vaccines, the cells being concentrated and intermixed in therequired proportions.

In general, B. vulgatus and F. necrophorum should be used in thevaccines in amounts of from 0.25 to 2 parts by weight on a dry cellbasis per part of T. hyodysenteriae. For the oral vaccine, at least oneof these bacteria should be present in the amount specified, and in apreferred embodiment, both of them are present in this amount. For theparenteral vaccine, the addition of only B. vulgatus is preferred.Killed cells of F. necrophorum may tend to produce undesirable sideeffects, such as abscesses, when injected intramuscularly orsubcutaneously.

On the basis of present information, it appears that the preferredproportions of the additional bacteria (B. vulgatus and F. necrophorum)are within the range from 0.5 to 1.5 parts by weight (dry basis) perpart of T. hyodysenteriae. Therefore, for the oral vaccine, at least oneand preferably both of the added bacteria will be present in thisamount, while the B. vulgatus cells will be used in this amount in theparenteral preparation.

Where the parenteral preparation is intended for intramuscularinjection, it is not believed to be beneficial to use an adjuvant.However, for subcutaneous injection an adjuvant may have some value. Forthis purpose a meat-animal acceptable adjuvant, such as aluminumhydroxide, can be added. For example, aluminum hydroxide can be used ata final concentration in the injectable preparation of from 0.25 to 1%Al₂ O₃.

The oral preparation should be enteric-coated. As used herein the term"enteric-coated" refers to a coating which is resistant to dissolving inthe swine stomach while dissolving in the swine intestines. As disclosedin the co-pending application Ser. No. 934812 of Robert A. Goodnow,filed on even date herewith, entitled "Oral Vaccine for Swine Dysenteryand Method of Use", such enteric coatings are preferably selected sothat they are insoluble in water at a pH below 5.0 while being slowlysoluble in water at a pH of 5.8 to 6.2.

Any of the known enteric coatings which meet these solubility or pHconditions can be utilized. One suitable coating material is celluloseacetate phthalate, which may be plasticized with diethyl or dibutylphthalate so that the coating is more resistant to cracking. Forapplication, the enteric coating material may be dissolved in a suitablevolatile organic solvent, and the enteric coat may be built up in aseries of applications to assure that the coating will be complete andrelatively uniform. One well known procedure of this kind is referred toas the Open-Pan Ladle Coating Process. For example, 30 to 40 parts byweight of cellulose acetate phthalate together with 8 to 10 parts ofdiethyl phthalate may be dissolved in 250 to 300 parts by weight ofacetone to form a coating solution for such application.

Suitable resins may also be used, such as acrylic resins prepared foruse as enteric coatings. One such product is sold under the trademark"Eudragit L 90" by Rohm Pharma Gmbh, Darmstadt, West Germany. Thisenteric coating material is supplied in granular form containing 10%water. The release pH of Eudragit L can be increased where desired bymixing it with Eudragit S. The manufacturer describes Eudragit L assoluble in intestinal juice from pH 6.0 and Eudragit S as soluble frompH 7.0. Eudragit L and mixtures with Eudragit S are soluble in ethanoland acetone, which may be used for applying the coating. They may beplasticized, if needed with various plasticizers, such as polyethyleneglycol, diethyl or dibutyl phthalate, triacetin, or castor oil. Forexample, from 70 to 90 parts by weight of Eudragit L can be combinedwith 20 to 30 parts by weight of diethyl phthalate. The Wurster CoatingProcess can also be used to apply the enteric coating. This process isdescribed in U.S. Pat. Nos. 3,241,520 and 3,253,944. It is carried outas a commercially available service by Coating Place, Inc., Verona,Wisconsin.

The enteric-coated oral vaccine is preferably in the form of pellets orgranules which can be readily mixed with swine feed material foradministration to the animals. For example, such granules may range fromabout -20 mesh to +100 mesh (U.S. Standard Screen). The granules aremixed with a finely-divided feed material such as a ground feed used forpigs after weaning. Any swine or pig feed material can be used, such asa basal ration containing ground corn, rolled oats, soybean meal,minerals and vitamins. The coated granules may also be premixed withvitamin-mineral fortification premixes, which are later combined withthe other feed ingredients.

To act as a filler or bulk stabilizer for desiccation and pelletpreparation, standard filler substances may be added to the cell slurrysuch as sucrose, dextrose, lactose, etc. In general, the amount offiller-stabilizer to be added may range from about 10 to 50 parts byweight of filler per 100 parts of cells (dry basis). Prior to theaddition of the filler, the cell concentrate preferably contains inexcess of 3.0 milligrams of cells (dry basis) per milliliter of slurry.For example, the cell concentrates may contain from about 4 to 7milligrams of cells (dry basis) per milliliter of cell slurry. Theparticular concentration is not critical, since most of the residualwater of the slurry is removed by a suitable drying procedure inpreparing the pellets.

The mixed cell slurry containing the added binder may be dried by asuitable biological drying procedure such as freeze-drying. Preferably,the drying is carried out at a relatively low temperature, such as below40° C. The dried material is then pulverized to a finely-dividedcondition for preparing tablets or granules.

For example, the mixed cell concentrates in the form of liquid slurriesare mixed with sucrose and cellulose, and kneaded to a doughyconsistency. The dough is then extruded in the form of noodles orribbons, which are broken up and formed into granules. The granule sizeis not critical, but desirably is of a size smaller than 20 mesh (U.S.Standard Screen). The granules are dried in an oven at a relatively lowtemperature, such as 37° C. until most of the moisture has been removed.The final moisture content is not critical, and desirably may range fromabout 1 to 3% water by weight.

In applying tablet and granule coatings, a dye may be included as acolorant for the coating. This permits the coating to be more readilyinspected for thickness and uniformity, and makes it easier to detectimperfections in the coatings. In practicing the present invention, itis desirable to use a dye in the enteric coatings of the presentinvention, although it is not essential with respect to the desiredimmunizing action. Suitable dyes include Lake Blue No. 2 and crystallineviolet dye.

While the vaccines of the present invention may be applied to adultswine, such as breeding sows, an important use is with growing pigs. Forexample, the method may be applied to feeder pigs, starting at the ageof about 3 to 8 weeks. The method may also be applied to older pigsduring their growth period prior to marketing. The pigs raised underfield conditions are highly subject to swine dysentery infection withconsequent economic loss due to lowering of the rate of weight gain andthe feed efficiency. By increasing the resistance of the pigs to swinedysentery infection, optimum rates of weight gain may be maintained.

In practicing the present invention, using either the oral vaccine, theparenteral vaccine, or both, the dose level can be related to the amountof T. hyodysenteriae administered. For example, with the oralpreparations, it is desirable to administer at least 3 milligrams of thekilled cells of T. hyodysenteriae (dry basis) per animal. Preferably,the doses are administered daily (once every 24 hours), such as byadmixture of the enteric-coated granules with a feed material, and thedosing is continued for a period of at least five days, such as from 5to 15 days. In a preferred embodiment, the oral doses contain at least 4mg. of the T. hyodysenteriae cells (dry basis) per dose, such as dosesin the range of 4 to 6 mg.

For the parenteral vaccine, it is preferred to inject at least 2milligrams (dry basis) of T. hyodysenteriae cells per animal per dose.If necessary, the parenteral dose can be repeated, such as a total offrom 2 to 3 doses. In a preferred embodiment, the parenteral dose maycontain from 3 to 6 mg. (dry basis) of T. hyodysenteriae cells per dose.

Where the same animals are to be given both the oral and parenteralpreparations, it is preferred to follow the dosing procedure describedin the co-pending application Ser. No. 935062 of Delbert L. Harris andRobert A. Goodnow, filed on even date herewith, and entitled "Method ofIncreasing the Effectiveness of Oral Vaccination for Swine Dysentery".However, the oral and parenteral vaccines of this invention can also beused separately, or with a different administration procedure thandescribed in the Harris and Goodnow application.

The invention is further illustrated by the following examples.

EXPERIMENTAL EXAMPLES Materials and Methods

Experimental Animals--Pigs from a herd with no history of swinedysentery were placed in isolation units at approximately 8 weeks of ageand fed a 16% protein grower ration which contained no drugs.

Preparation of Inoculum--Cultures of Treponema hyodysenteriae (isolateB204) were grown approximately 24 hours in aerobically preparedtrypticase soy broth containing 10% fetal calf serum under deoxygenatedH₂ :CO₂ at 38° C. One hundred ml of whole culture was administered toeach pig via stomach tube following a 48 hour starvation period. Theisolate of T. hyodysenteriae had not been passaged more than 15 times invitro.

Preparation of Vaccines--An oral vaccine and a parenteral vaccine wereprepared containing Treponema hyodysenteriae isolate (B204, No. 31212)and Bacteroides vulgatus (Strain 28, ATCC No. 31376).

A. preparation of Treponema hyodysenteriae Antigen: Growth:

The T. hyodysenteriae organism was grown in trypticase soy brothenriched with 10% fetal calf serum (Gibco, Grand Island, N. Y.) in a28-liter pilot New Brunswick fermenter and grown to an average cell masslevel of equal to or greater than 2.0×10⁹ cells/ml. Seventy-two (72)liters of T. hyodysenteriae grown to 1.25 milligrams dry wt. antigen/mlof fermenter culture was used in this preparation.

Concentration:

The cell culture was inactivated with a 1:10,000 concentrationMerthiolate. The cell mass was concentrated by forcing the cell massthrough a 100,000 molecular weight size membrane by positive airpressure. The cell slurry was concentrated to a level of 30 milligramsdry wt. antigen/ml per 3.5 liters of slurry.

B. preparation of Bacteroides vulgatus Antigen:

The B. vulgatus organism was grown in standard peptone yeast extractglucose medium to a level of 2.3 milligrams dry wt. antigen/ml fermentergrown culture. Forty-five (45) liters of this cell culture wasconcentrated by similar ultrafiltration to a cell mass level of 34.7milligrams dry wt. antigen/ml per 3 liters slurry.

C. preparation of Combination Antigens:

The 3.5 liters of T. hyodysenteriae slurry and 3.0 liters of B. vulgatuswere combined in a common vessel. This slurry was concentrated bysimilar ultrafiltration to 2.85 liters of slurry.

D. preparation of Enteric Coat on Vaccine:

Carrier:

Twenty (20) mesh sucrose pariels were sprayed with the bacterial antigenslurry to a 15.97% antigen/product wt. increase. Drying temperatureswere maintained between 102°-110° F. above the drying bed and between144°-165° F. on the inlet slurry line. After the antigen was coated uponthe sugar needs, an enteric resin overcoat was sprayed on theantigen-sugar needs.

E. entric Solution:

83.3 Grams Eudragit L90, and 25 grams of diethyl phthalate was added to1.0 liters of methanol. This solution was then q.s. to 1.67 liters withacetone. This solution was sprayed on the sugar-antigen carrier and airdried according to the Wurster Process. See U.S. Pat. Nos. 3,253,944 and3,241,520. The Wurster coating process was performed by Coating Place,Inc., Verona, Wisconsin. One (1) liter of concentrated slurry resultedin 1 kilogram of oral swine dysentery vaccine.

Experiment 1

Thirty-four pigs were randomly assigned to individual isolation units.The pigs were immunized as follows:

    ______________________________________                                        No. of     Parenteral Vaccine                                                 Group  Pigs    Intramuscular                                                                            Intraperitoneal                                                                         Oral Vaccine                              ______________________________________                                        I      9       +          -         -                                         II     9       +          -         +                                         III    8       -          +         +                                         IV     8       -          -         -                                         ______________________________________                                    

On day 0 the pigs were weighed individually and pigs in groups I, II,and III were injected with 5 ml/pig of the parenteral vaccine. Startingon day 9, the oral vaccine was fed to the pigs in groups II and III bymixing 1.0 gram of the vaccine in approximately 500 grams of feed whichwas spred across the floor for consumption by the pigs. Additional feedcontaining no vaccine was given later. The oral vaccine was administereddaily through day 23. On day 14, the pigs in groups I, II, and III wereinjected with 5 ml/pig of the parenteral vaccine. On day 24, all pigswere weighed. Feed with withheld on days 24 and 25. On day 26, rectalswabs were collected and all pigs were challenged with T. hyodysenteriae(8.0×10⁹ organisms per pig).

Experiment 2

Forty-five pigs were randomly assigned to isolation units. The pigs wereimmunized as follows:

    ______________________________________                                               No. of   Parenteral Vaccine                                            Group  Pigs     Intramuscular  Oral Vaccine                                   ______________________________________                                        I      8           +           -                                              II     9           -           +*                                             III    9           +           +*                                             IV     10          +           +* (continuous)                                V      9           C**         C**                                            ______________________________________                                         *Group II and III received oral vaccine daily until challenge;                Group IV received oral vaccine daily before and after challenge.              **Received control placebo-no T. hyodysenteriae or B. vulgatus antigen.  

On day 0 the pigs were weighed individually and pigs in groups I, III,and IV were injected with 5 ml/pig of parenteral vaccine. On day 8 theoral vaccine was initiated in groups II, III, and IV as described above.The oral vaccine was continued through day 28 for groups II and III. Onday 15 the pigs in groups I, III, and IV were injected with 5 ml/pig ofparenteral vaccine. On day 28 all pigs were weighed. Feed was withheldon days 29 and 30. On day 31, rectal swabs were collected and all pigswere challenged with T. hyodysenteriae (9.0×10⁹ organisms per pig).

Pigs were weighed at weekly intervals after challenge and rectal swabswere collected twice weekly. Clinical evaluation of response tochallenge was recorded for each pig on a daily basis for 34 dayspost-inoculation (DPI).

Evaluation of Response to Challenge--Each pig was observed daily and 3clinical parameters were scored on a scale of 1 to 4.

General Condition: 1=normal; 2=gaunt, mildly inactive; 3=very gaunt,rough hair, very inactive; 4=emaciated, moribund.

Feces Consistency: 1=normal, firm; 2=soft, not formed; 3=liquid;4=watery.

Feces Composition: 1=normal; 2=increased mucus; 3=increased mucus, smallflecks of blood; 4=large amount of blood present.

For purposes of tabulation, a score of 3 or 4 was considered to berespectively cachexia, diarrhea and dysentery in the 3 parametersobserved.

A daily index for each pig was calculated as the mean of the scores forthe 3 individual observations. Pigs that died were given the maximumscore of 4 for each of the parameters through the remainer of the studyfor purposes of analysis.

Isolation of T. hyodysenteriae--A rectal swab was collected and placedin a culturette holder containing transport medium. The swab was held at4° C. and inoculated onto selective medium for isolation of T.hyodysenteriae within 24 hours of collection. The selective mediumcontained trypticase soy agar, 5% bovine blood, and 400 mcg/ml ofSpectinomycin sulfate and was incubated under 80% H₂ and 20% CO₂ withpalladium catalysts at 42° C. The selective medium was examined at 2, 4,and 6 days of incubation and the presence or absence of pathogenic (βhemolytic) T. hyodysenteriae was recorded.

Necropsy Procedures--A necropsy was performed on all pigs that diedduring the trial. Macroscopic lesions were recorded and a swab wascollected from the colonic mucosa for isolation of T. hyodysenteriae.Salmonella isolations were attempted from the mesenteric lymph node,small intestine and colon.

The data is set out below in Tables A, B, and C for Experiment 1 andTables D, E, and F for Experiment 2.

Results and Discussion

Exp. 1--Some pigs in all vaccinated groups (I, II, and III) showedsymptoms of diarrhea and dysentery but of much less severity andduration than nonvaccinated control pigs (group IV). The incubationperiod of the disease was delayed in vaccinated pigs as compared tononvaccinated pigs. Vaccinated pigs gained weight more rapidly andgained more total weight than nonvaccinated pigs.

Treponema hyodysenteriae were isolated from the feces of pigs in thenonvaccinated groups within 4 days post inoculation. By contrast, mostvaccinated pigs did not shed T. hyodysenteriae until 14 days postinoculation. Vaccination did not stop the establishment of infection byT. hyodysenteriae.

One pig died in the nonvaccinated group due to swine dysentery. No pigsdied in the vaccinated groups.

Exp. 2--The pigs in this study appeared to be more severely challengedthan Exp. 1. Pigs in group III had less clinical signs of swinedysentery and performed better based on weight gains than pigs in groupV (controls). Pigs in group IV were severely affected very early afterchallenge which may have been due to the continual exposure to the oralvaccine. By contrast, this was the only group in which no deathsoccurred.

Seven pigs died during the study. Six of these pigs died of swinedysentery while one pig in group II died of necroproliferativeenteritis.

                  TABLE A                                                         ______________________________________                                        Clinical Responses in Pigs Inoculated Intragastrically                        with T. hyodysenteriae Exp. 1                                                            Group                                                                           I        II       III    IV                                      Clinical Response                                                                          N=9.sup.a                                                                              N=9      N=8    N=8                                     ______________________________________                                        Diarrhea:                                                                     Day of Onset.sup.b                                                                         15.33    25.00    21.63  12.25                                   Days Duration                                                                              6.67     2.78     2.88   15.13                                   No. Affected 6        6        5      8                                       Dysentery:                                                                    Day of Onset 21.33    25.22    24.00  14.00                                   Days Duration                                                                              4.89     1.11     1.13   9.63                                    No. Affected 5        5        4      7                                       Cachexia:                                                                     Day of Onset 29.00    34.00    34.00  18.63                                   Days Duration                                                                              1.78     0        0      5.50                                    No. Deaths   0        0        0      1                                       Combined Index                                                                             1.46     1.25     1.27   1.97                                    ______________________________________                                         .sup.a N equals number of pigs per group                                      .sup.b Study terminated at 34 days. Calculations are based on a value of      34 assigned to each pig which remained normal.                           

                  TABLE B                                                         ______________________________________                                        Cumulative Average Gain (pounds) per Pig Inoculated                           Intragastrically with T. hyodysenteriae Exp. 1                                Days        Group                                                             Post inoculation                                                                          I        II       III    IV                                       ______________________________________                                         7          6.66     6.00     5.75   3.00                                     14          11.33    15.00    12.25  .25                                      21          20.11    23.11    20.25  9.36                                     28          29.33    28.45    26.12  18.07                                    35          40.00    39.56    35.75  25.07                                    ______________________________________                                    

                  TABLE C                                                         ______________________________________                                        Isolation of Pathogenic T. hyodysenteriae from the Feces                      of Pigs Inoculated Intragastrically with T. hyodysenteriae Exp. 1             Days        Group                                                             Post inoculation                                                                          I        II       III    IV                                       ______________________________________                                         0           0/9.sup.a                                                                             0/8      0/8    0/7                                       4          0/9      0/9      0/8    2/8                                       7          4/9      1/9      2/8    4/8                                      11          7/9      8/9      3/8    8/8                                      14          8/9      8/9      6/8    7/7                                      21          6/9      5/7      5/8    6/7                                      28          7/8      5/9      5/8    5/7                                      ______________________________________                                         .sup.a Denominator equals number of pigs sampled. Numerator equals number     of pigs positive.                                                        

                  TABLE D                                                         ______________________________________                                        Clinical Responses in Pigs Inoculated Intragastrically                        with T. hyodysenteriae Exp. 2                                                           Group                                                                           I        II      III   IV    V                                    Clinical Response                                                                         N=8.sup.a                                                                              N=9     N=9   N=10  N=9                                  ______________________________________                                        Diarrhea:                                                                     Day of Onset.sup.b                                                                        16.00    12.33   21.33 9.0   12.78                                Days Duration                                                                             9.63     4.78    3.56  8.2   10.22                                No. Affected                                                                              7        8       8     10    9                                    Dysentery:                                                                    Day of Onset                                                                              16.00    13.22   21.78 8.9   13.00                                Days Duration                                                                             6.38     4.78    2.44  6.5   8.67                                 No. Affected                                                                              7        8       8     10    9                                    Cachexia:                                                                     Day of Onset                                                                              25.88    14.78   27.22 17.7  17.11                                Days Duration                                                                             6.13     7.44    1.44  4.1   7.22                                 No. Deaths  1        2       2     0     2                                    Combined Index                                                                            1.78     1.64    1.39  1.64  1.91                                 ______________________________________                                         .sup.a N equals number of pigs per group                                      .sup.b Study terminated at 35 days. Calculations are based on a value of      35 assigned to each pig which remained normal.                           

                  TABLE E                                                         ______________________________________                                        Cumulative Average Gain (pounds) per Pig Inoculated                           Intragastrically with T. hyodysenteriae Exp. 2                                Days        Group                                                             Post inoculation                                                                          I       II      III   IV    V                                     ______________________________________                                         7          -.12    -1.00   6.22  3.10  3.56                                  14          2.25    1.53    5.89  -5.60 5.28                                  21          4.68    3.03    10.00 .10   1.35                                  28          11.54   6.36    17.00 5.90  7.07                                  35          16.68   18.50   21.97 11.80 13.64                                 ______________________________________                                    

                  TABLE F                                                         ______________________________________                                        Isolation of Pathogenic T. hyodysenteriae from the Feces                      of Pigs Inoculated Intragastrically with T. hyodysenteriae Exp. 2             Days         Group                                                            Post inoculation                                                                           I       II      III   IV    V                                    ______________________________________                                         0            0/8.sup.a                                                                            0/9     0/9   0/10  0/9                                   4           2/8     1/9     1/9   3/10  0/9                                   7           6/8     4/9     0/9   9/10  5/9                                  11           2/8     6/8     5/9   9/10  7/9                                  14           4/8     5/8     3/9   8/10  6/8                                  18           3/7     6/8     2/8   6/10  6/7                                  21           2/7     6/8     4/8   5/10  4/7                                  25           3/7     3/7     3/8   1/10  2/7                                  28           2/7     4/7     4/8   0/10  4/7                                  32           0/7     0/7     0/7   0/10  0/7                                  35           0/7     2/7     0/7   0/10  0/7                                  ______________________________________                                         .sup.a Denominator equals number of pigs sampled. Numerator equals number     of pigs positive.                                                        

FURTHER EXAMPLES Oral Preparation

Treponema hyodysenteriae, Bacteroides vulgatus and Fusobacteriumnecrophorum are each separately grown under anaerobic conditions, aspreviously described, to provide immunizing antigens for use incombination. Each culture after Merthiolate inactivation, is mixedtogether and concentrated by ultrafiltration to provide at least 30mg/ml T. hyodysenteriae, 34.7 mg/ml Bacteroides vulgatus and 35.0 mg/mlFusobacterium necrophorum in the wet mixed slurry. Enteric coatedgranules are then prepared.

The concentrated mixed antigen slurry is combined with the otheringredients in the following proportions:

1500 cc antigen slurry

11.5 kilo sucrose

3.5 kilo microcrystalline cellulose

0.2% dry weight Lake Blue No. 2 Dye

H₂ o added as needed for obtained proper texture

Once this mixture is partially mixed the moistened mass of material isrun through a commercial extruder at least three times to provide auniform mix of antigen to carrier. The cylindrical pieces are thenshaped into uniform beads in a manumerizer. The bead preparation isdried at least 1-8 hours leaving 1-3% moisture content. An entericcoating is then applied to the beads by either the Wurster or anOpen-Pan Ladle type coating process. The preferred coating is preparedfrom 15 parts by weight Eudragit S 90 (90% active, 10% water) and 85parts of Eudragit L 90 (90% active, 10% water). This mixture may bedissolved in ethanol for application to the vaccine granules. Itprovides a coating of increased resistance to dissolving in the stomachwhen the pigs are fed continuously. (Eudragit S 90 and L 90 are sold byRohm Pharma Gmbh, Darmstadt, West Germany.)

Parenteral Preparations

Cultures of T. hyodysenteriae and B. vulgatus are prepared as previouslydescribed, and the cells are inactivated. The two cultures are mixed andconcentrated by ultrafiltration to provide at least 30 mg/ml T.hyodysenteriae and 34.7 mg/ml B. vulgatus. A preparation forintramuscular injection is prepared by diluting the combinedconcentrated slurry with sterile water to obtain an injectablepreparation containing approximately 1 mg/ml of each antigen (drybasis). In one administration procedure, 5 ml of this dilutedpreparation is injected intramuscularly into each pig, comprising a doseof 5 mg of each antigen per pig. After two weeks, each pig is givenanother injection of the same combined intramuscular preparation.

For subcutaneous administration, the diluted intramuscular preparationcontaining 1 mg/ml of each antigen is combined with an aluminumhydroxide adjuvant. One part by volume of the adjuvant as a 2% aqueoussuspension (aluminum oxide basis) is added per five parts by volume ofthe diluted antigens. This preparation is administered in 6 ml doses perpig, providing 5 mg of each antigen per dose. The preparation isinjected subcutaneously, and a second dose is administered after 14days. If desired, the amount of adjuvant can be increased to a level of0.5 to 1% Al₂ O₃.

We claim:
 1. An oral preparation for increasing the resistance of swineto dysentery infection, comprising enteric-coated orally-administrablepellets containing concentrated killed cells of a virulent isolate ofTreponema hyodysenteriae in combination with concentrated killed cellsof other bacteria selected from the group consisting of Bacteroidesvulgatus, Fusobacterium necrophorum, and mixtures thereof, at least oneof said other bacteria being present in an amount of from 0.25 to 2parts by weight (dry basis) per part of said Treponema hyodysenteriae.2. The oral preparation of claim 1 in which said other bacteria isBacteroides vulgatus.
 3. The oral preparation of claim 1 in which saidother bacteria is Fusobacterium necrophorum.
 4. The oral preparation ofclaim 1 in which said other bacteria comprises both said Bacteroidesvulgatus and Fusobacterium necrophorum, and each are present in anamount of from 0.25 to 2 parts by weight (dry basis) per part of saidTreponema hyodysenteriae.
 5. The oral preparation of claim 1 in whicheach of said other bacteria that is present are present in an amount of0.5 to 1.5 parts by weight (dry basis) per part of Treponemahyodysenteriae.
 6. The method of increasing the resistance offield-raised swine to dysentery infection, comprising orallyadministering to the swine prior to their contracting the infection aplurality of doses of enteric-coated pellets containing concentratedkilled cells of Treponema hyodysenteriae in combination withconcentrated killed cells of other bacteria selected from the groupconsisting of Bacteroides vulgatus, Fusobacterium necrophorum, andmixtures thereof, each of said doses providing at least 3 milligrams(dry basis) of said Treponema hyodysenteriae cells together with from0.25 to 2 parts by weight (dry basis) of at least one of said otherbacteria.
 7. The method of claim 6 in which said other bacteria isBacteroides vulgatus.
 8. The method of claim 6 in which said bacteria isFusobacterium necrophorum.
 9. The method of claim 6 in which said otherbacteria comprises both said Bacteroides vulgatus and Fusobacteriumnecrophorum, and each are present in an amount of from 0.25 to 2 partsby weight (dry basis) per part of said Treponema hyodysenteriae.
 10. Themethod of claim 6 in which each of said other bacteria that is presentis present in an amount of 0.5 to 1.5 parts by weight (dry basis) perpart of Treponema hyodysenteriae.